Anal. Chem. 1995, 67, 3746-3752
DuallAnalyte Fiberloptic Sensor for the Simultaneous and Continuous Measurement of Glucose and Oxygen Lin Li and David R. Walt* Max Tishler Laboratory for Organic chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155
A fiber-opticsensor for the continuous and simultaneous determination of glucose and oxygen is described. The sensor is comprised of dual-analyte sensing sites in defined positions on the distal end of an imaging fiber (350pm 0.d.). Each sensing site is an individual polymer cone covalently attached to the activated fiber surface using localized photopolymerization. The oxygen sensor consists of a double-layer polymer cone. The inner polymer cone is a hydrophobic gas-permeable copolymer containing an oxygen-sensitive ruthenium dye, and the outer layer is a poly(hydroxyethy1methacrylate) (HEMA) polymer. The glucose sensor is an oxygen sensor with a poly-HEMA outer layer containing immobilized glucose oxidase. The fluorescence images of both sensing sites are captured with a CCD camera, and the measured fluorescence intensities are related to analyte concentrations. Oxygen quenching data for both sensing sites fit a two-siteStern-Volmer quenching model. The sensor has been used to simultaneouslymonitor independent changes in glucose and oxygen concentrations. Glucose calibration curves were obtained under varying oxygen tensions, and the detection limit is 0.6 mM glucose. The effect of fluctuations in oxygen partial pressure on the glucose response can be used to calibrate the sensor. The sensor response time varies from 9 to 28 s, depending on the different thicknesses of the enzyme layer. The sensor maintains the same sensitivity for 2 days. Multiple glucose sensing sites with different enzymatic activities can be immobilized on the distal end of the fiber, affording control of the linear range.
be caused by changes in oxygen tension during synaptic events6 In addition, the sensor's in vivo performance can be affected by the availability of local oxygen, which varies due to the uneven distribution of oxygen within the tissue and the degree of tissue damage caused by sensor implantati0n.~>8 Oxygen-independentglucose biosensors have been developed in order to overcome these problems. Glucose enzyme electrodes have been described which use mediators or redox polymers instead of oxygen to transfer electrons between the reduced enzyme and an electrode s u r f a ~ e . ~ -Such ' ~ sensors are much less sensitive to oxygen, but the oxygen dependence has not been eliminated completely. An alternative approach is to employ glucose sensors in which a constant oxygen supply is provided internally. A glucose microsensor has been fabricated by locating the sensing surface in an oxygenated microenvironment therefore, the glucose measurement is independent of the oxygen concentration in the ~amp1e.l~Other newly designed glucose sensors are based on an unlimited oxygen supply from the atm~sphere.~J~ Many of these sensors are not optimal for either in vivo or in vitro glucose monitoring due to incompatible sensor sizes or complicated configurations. For some applications, the concentration of oxygen must be known in order to understand biological events involving g l u ~ o s e . ' ~ ~ ' ~ We have developed a novel technique for fabricating compact multianalyte fiber-optic chemical sensor^.^^-^^ Multiple analyte sensing sites are placed in precise positions using localized photopolymerization of appropriate dye indicators or enzymes on the distal end of an optical imaging fiber. The changes in the optical properties at each site are transmitted through an imaging fiber through distinct optical pathways and simultaneously moni(6) Zimmerman, J. B.; Wightman, R M. Anal. Chem. 1991,63, 24-28.
Glucose biosensors have been utilized widely in clinical analysis, biomedical research, and biote~hnology.'-~In most cases, the 'sensing scheme is based on the enzymatic oxidation of glucose by glucose oxidase. Glucose can be monitored by analyzing either the reaction products or the oxygen consumption. The response of these sensors depends on the local oxygen concentration, which limits their application. For example, in neurochemical applications, fluctuations in sensor response can (1) Michael, A C.; Justice, J., Jr. Anal. Chem. 1987,59, 405-410. (2) Dermal, B. A. A; 11, S.-Y.;Schmid, R D. Biosens. Bioelectron. 1992,7, 133139. (3) Bindra, D. S.; Zhang, Y. N.; Wilson, G. S.; Stemberg, R; Thevenot, D. R.; Moatti, D.; Reach, G. Anal. Chem. 1991,63, 1692-1696. (4) Amine, A; Pabiarche, G. J.; Marrazza, G.; Mascini, M. Anal. Chim. Acta 1991,242,91-98. (5) Rishpon, J.; Shabtai, Y.; Rosen, I.; Zibenberg, Y.; Tor, R.; Freeman, A Biotechnol. Bioeng. 1990,35, 103-107.
3746 Analytical Chemistry, Vol. 67, No. 20, October 75, 7995
(7) Zhang, Y.; Wilson, G. S. Anal. Chim. Acta 1993,281, 513-520. (8)Brunstein, E.; Abel, P.; Gens, A; Eich, K; Woedtke, T. V. Biomed.Biochim. Acta 1989,48,911-917. (9) Cass. A E. G.; Davis, G.; Francis, G. D.; Hill, H. A 0.;Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A P. F. Anal. Chem. 1984,56, 667-671. (10) Yokoyama, K; Tamiya, E.; Karube, I. Anal. Lett. 1989,22, 2949-2959. (11) Gregg, B. A.; Heller, A Anal. Chem. 1990,62, 258-263. (12) Pishko, M. V.; Michael, A C.; Heller. A Anal. Chem. 1991,63, 22682272. (13) Cronenberg, C.; Groen, B. V.; Beer, D. D.; Heuvel, H. V. D. Anal. Chim. Acta 1991,242, 275-278. (14) Kusano, H. Clin. Phys. Physiol. Meas. 1989,10,1-9. (15) Casciari, J. J.; Sotirchos, S. V.; Sutherland, R M.J. Cell. Physiol. 1992,151, 386-394. (16) Zinker, D.; Namdaran, K; Wilson, R; Dracy, D.; Lacy, D.; Wassaerman, D. Diabetes 1993,42,A44. (17) Bamard, S. M.; Walt, D. R. Nature 1991,353, 338-340. (18) Walt, D. R; Agayn, V.; Bronk, K; Branard, S. Appl. Biochem. Biotechnol. 1993,41,129-137. (19) Bronk, K S.; Walt, D. R. Anal. Chem. 1994,66, 3519-3520.
0003-2700/95/0367-3746$9.0010 0 1995 American Chemical Society
tored with a chargedcoupled device (CCD) camera. This strategy is used here to develop a dual-analytefiber-optic sensor for glucose and oxygen. The oxygen sensing site consists of an oxygensensitive ruthenium complex in a hydrophobic gas-permeable copolymer on the distal end of an imaging fiber. The glucose sensing site is composed of a second oxygen sensing polymer cone coated with polyfiydroxyethyl methacrylate) (HEM4 containing immobilized glucose oxidase. The concentrations of glucose and oxygen are proportional to the changes in the fluorescence intensities of the ruthenium complex at each sensing site. The response characteristics of this dual-analyte sensor for glucose and oxygen are presented. EXPERIMENTAL SECTION Materials. Tris(2,2'-bipyridyl (bpy))ruthenium(II) chloride, tris (phenanthroline (phen))ruthenium(IDchloride, benzoin ethyl ether (BEE), and a-D-glucose were all purchased from Aldrich Chemical Co. (Milwaukee, WI). Glucose oxidase @?-D-Glucose, oxygen 1-oxidoreductase; EC 1.1.3.4) (162 units/mg type VII, Aspelgillus niger) and fumed silica (0.014 pm 0.d.) were obtained from Sigma Chemical Co. (St Louis, MO). (Acry1oxy)propylmethylsiloxane (15-20%) and dimethylsiloxane (80-85%) copolymer (ps802) were obtained from Gelest, Inc. (Tullytom, PA). Ethylene glycol dimethacrylate ( E G D W and 2-hydroxyethylmethacrylate (HEMA) were obtained from Polysciences Inc. (Warrington, PA). Ru(4,7diphenyl(Fh)-l,l@phen)3(Cl)~ was synthesized and puritied as described All the other reagents were used as received. Oxygen and nitrogen were purchased from Northeast Airgas, Inc. (Salem, NH). Solutions of varying oxygen tension were prepared by bubbling 0.1 M pH 7.4 phosphate buffers with appropriate gas for at least 15 min. All reagent solutions were prepared with distilled, deionized water purified with a Barnstead Nanopure system. Glucose solutions were prepared in 0.1 M pH 7.4 phosphate buffer and stored in the refrigerator for 24 h before use to allow equilibration between a-&glucose and @-glucose. The immobilization of Ru(bp~)3~+ or Ru(phen)32+on fumed silica (0.014 pm 0.d.) was done as follows. Initially, 30 mg of Ru0 dye was added to 60 mg/mL fumed silica/methylene chloride solution. The solution was stirred overnight to adsorb the Ru(II) complex on silica. The silica particles containii Ru(II) dye were washed with 150 mL of methanol and dried in an oven at 135 "C. The Ru(II)/silica particles were stored in the dark at room temperature. The singlecore stepindex hard clad silica (HCS) fibers (750 pm o.d., 550 pm i.d.) were purchased from Ensign-BickfordOptics Co. (Avon, CT). S i c a imaging fibers with an outer core diameter of 350 pm, containing 6OOO sensing elements, were purchased from Sumitomo Electric U.S.A., Inc. (Torrance, CA). All fibers were polished before use on lapping 6lms purchased from General Fiber Optics, Inc. (Fairfield, NJ). Fabrication of Oxygen Sensor. An oxygen sensor was prepared by immobilizing an oxygen-sensitive Ru (ID complex in a gas-permeable siloxane polymer on the distal end of a single core silica fiber using photopolymerization. These sensors were used to examine the optimal experimental conditions. The photopolymerization approaches and procedures have been described previously.21 The oxygen-sensitivecopolymer solution was (20) Watts, R J.; Crosby, G. AI. An.Chem. Soc. 1971,93, 3184-3188. (21) Barnard, S. M. Ph.D.Thesis, Tufts University, Medford, MA, 1992.
prepared by dissolving 30 mg of BEE in 0.25 mL of methylene chloride solution containing varying amounts of Ru(ID complex and 0.5 mL of ps802 siloxane copolymer. Fabrication of Glucose and Oxygen Sensing Arrays. The imaging fiber was silanized in a 10%(v/v) 3-(trimethoxysilyl)propyl methacrylate/acetone solution to activate the fiber surface before use. The instrumentation of the photodeposition system has been detailed previou~ly.'~Briefly, the oxygen sensing cone was formed at a precise location on the distal end of the fiber by siteselectively photopolymerizing the oxygen-sensitive poly (dimethylsiloxane) copolymer. The copolymer solution was prepared as described above with 1mg/mL Ru(€'h~phen)3~+/methylene chle ride stock solution. Excitation light (wavelength, 350 f 80 nm) from a xenon-mercury arc lamp was focused through a 400 pm diameter pinhole and a 15x reflective objective onto the proximal end of the fiber. The light was transmitted to the other end of the fiber in a 27 pm diameter circular area of the fiber. An electronic shutter was used to control the light illumination time on the fiber surface. The distal end of the fiber was dipped into the oxygen-sensitivepolymer solution and removed, leaving a drop on the surface. The electronic shutter was then opened for 3 s to initiate photopolymerization on the fiber surface. The oxygen sensing polymer cone was formed only at the illuminated area. The distal end of the fiber was rinsed with ethanol to remove excess monomer solution. After polymerization was complete, the fiber was repositioned for a subsequent polymerization. Four siloxane polymer cones were photodeposited on the distal end. The glucose sensing cone was prepared by depositing HEMA polymer containing glucose oxidase on the surface of the oxygensensitive siloxane polymer cone. The immobilization of glucose oxidase in HEMA polymer was unsuccessful when the enzyme was dissolved in an aqueous HEMA polymerization solution. Therefore, the following procedure was used, which was found to be effective for enzyme immobilization in poly-HEMA.22 Glucose oxidase (2 mg)was suspended in 0.5 mL of a 10%dextran solution and lyophilized. The dry particles of lyophilized enzyme were stored in a desiccator at 5 "C. The lyophilized glucose oxidase was suspended in 0.5 mL of a HEMA polymerization solution containiig 4% EGDMA cross-linker and 30 mg of BEE photoinitiator. S i c e photopolymerization is inhibited by oxygen, the solution was deoxygenated by bubbling with nitrogen for 15 min before use. A 100 pL aliquot of this monomer solution containii glucose oxidase was first illuminated for 60 s with 366 nm light to form a viscous oligomer. The distal end of the fiber was then immersed in this oligomer solution. A pinhole with a diameter of 800 pm (giving a focused spot with a diameter of 54 pm) was used to allow enough light to illuminate the entire oxygen polymer sensing cone. The fiber was illuminated for 15 s, and glucose oxidasecontaining HEMA was poeerized on the surface of the oxygen cone. The distal end of the fiber was rinsed with ethanol until the excess monomer solution was washed off the fiber. Generally, 30 s illumination is required to deposit a HEMA polymer on the imaging fiber without the prepolymerization step. The purpose of prepolymerizing the HEMA monomer solution before photodeposition is to minimize the light exposure time of the oxygen sensing cone, since the Ru(II) dye is photolabile. The second glucose sensing polymer cone was deposited using the same procedure as the first one but with a 14 s illumination time on the fiber surface. The remaining two oxygen sensing (22) Healey, B. G.; Walt, D. R, Tufts University, unpublished results.
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Imaging Fiber
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HEMA wlo Glucose Oxidase HEMA wl Glucose Oxidase Oxygen Sensing Site Glucose Sensing Site Schematic showing the cross-sectional view of the glucose and oxygen sensing sites. Figure 1.
cones were coated with a HEMApolymer without glucose oxidase using the same procedure as that used forthe first glucose sensing cone. The exlra layer of HEMA hydrogel provides better resiliency than ps802 siloxane polymer. All the sensors were soaked in pH 7.4 phosphate buffer for 2 h before use. Figure 1 shows schematically the cross section of oxygen and glucose sensing.sites on the distal end of an imaging fiber. Sensor Response Measurements. The oxygen sensor response on the singl-ore HCS silica fiber was monitored on a doublemonochromator fluorescence system.Z3 The excitation wavelength was 480 nm, and the emission wavelengths were 590, 600, and 610 for R~(bpy)3~+, R~(phen)3~+, and R~(€'h2phen)3~+, respectively. The sensor response at different oxygen concentrations was monitored until steadystate signals were obtained. The imaging sensor response was measured with use of a modifiedfluorescencemicroscope. The instrumentation has been described el~ewhere.'~The distal end of the fiber was immersed in an appropriate test solution, and the images of the distal end of the optical fibers were captnred by a CCD camera. The excitation and emission filters were 470 k 35 and ?600 nm, respectively. The fluorescence intensities at the sensing cones were obtained as a function of time by analyzing the captured images using appropriate imaging processing software (IPlab, Signal Analytics, Vienna, VA). The fluorescence intensities of sensing cones reported at different analyte concentrations were steadystate signals. For the stability experiment, the sensor was stored in pH 7.4 phosphate buffer in the dark at 4 "C overnight. RESULTSAND DISCUSSION
The placement of both glucose and oxygen sensors on the same optical fiber provides this biosensor array with the ability to measure both enzyme substrates simultaneously. Therefore, variations in oxygen do not compromise sensor performance. Sensor preparation involves a photopolymerization technique. Fmt, oxygen-sensitive sensors are deposited in a hydrophobic siloxane polymer. Next, an enzyme-containinghydrogel layer is photodeposited on top of some of the oxygen sensors. The resulting sensors are -50pm in diameter. The ability to precisely control the position and archikture of each sensing layer provides an e n 0 into a wide range of sensor and biosensor arrays. Oxygen Sensor. The oxygen sensitivity of each polymer sensing cone directly influences the performance of the dual(23) Walt, D. R Gabor, G.;Goyet, C. Annl Ckim. Acta 1993.274,47-52.
3748 Analytical Chemisty, Vol. 67, No. 20,October 15, 1995
analyte sensor. Several key parameters have been investigated to optimiie the response properties of oxygen sensors based on fluorescence quenching of R u m complexes under our experimental conditions. These parameters include the polymer sup port, the coordinating ligands a) of the Ru"f.L)3 complex, the photopolymerization time, and the amount of dye in the polymer matrix. The magnitude of the oxygen sensor response in pH 7.4 phosphate buffer was dependent on the photopolymerization time. By increasing the photopolymerization time from 5 to 10 s, a 50% decrease in the sensor fluorescenceintensitieswas observed (data not shown). Ru(II) complexes in siloxane polymers decompose upon irradiation with visible or UV light. Therefore, increasing the light exposure time of the RuUD complex during sensor fabrication caused more decomposition and diminished the fluorescence intensity. A photopolymerization time of 3 s was used for oxygen sensor fabrication. The dye loading in the siloxane polymer also innuenced the magnitude of the sensor response and its sensitivity. The fluorescence signal of the oxygen sensor increased with increasing concentrations of RuQI) complex in the siloxane monomer solution. However, more dye did not produce better sensitivity. The optimal dye loading for the highest sensitivity was as follows: Ru(bpy)~z+-silica/sioxane of 20 m g l m l R~(phen)3~+silica/siIoxane of 32 mg/mI, and Ru(Ph2phen)2+/siloxaneof 0.33 mg/mL in the siloxane copolymer solutions. Further increases in the dye concentration resulted in larger background signals and lower sensitivity. Immobilization of the Ru(II) complex on silica resulted in a heterogeneous medium for oxygen quenching, and a linear Stern-Volmer plot was obtained. Ru(F'h~phen)3~+ was not adsorbed on silica due to the limited amount of this commercially unavailable dye. The inrluences of polymer supports and coordinating ligands on the quenching of Rum complexes by oxygen have been studied in detail by Demas et al.",25 Under the present experimental conditions, the best oxygen-sensitive dye for the sensor was found to be Ru(€'h2phen)32+ in the ps802 siloxane polymer. Ru(€'hZphen)?+ has the longest excited-state lifetime and the largest solution Stem-Vohner quenching constant The oxygen sensor based on this complex is extremely sensitive to oxygen." F i r e 2 shows that this dye is 4 times more sensitive than either R u ( b p ~ ) 3 ~or + Ru(phen)3z+.The sensitivityobserved here is lower than that reported by Demas. The loss of sensitivity is caused mainly by the polymer used and photolysis of the Ru(II) complex during sensor fabrication. As a result, Ru(€'h~phen)3~+ was used exclusiveiy to prepare all the sensors described below. Glucose and Oxygen Sensing Array. Figure 3 shows the fluorescence image of a four-spot sensor captured in a pH 7.4 airsaturated phosphate buffer. Spots 1and 2 are glucose sensitive; spots 3 and 4 are oxygen sensitive only. Glucose sensing cone 1 was prepared with a longer photopolymerization time than that for cone 2, resulting in a larger polymer cone and thus more glucose oxidase immobilized in the polymer matrix. At the oxygen sensing sites, the RuUn complex fluorescence intensity is related to both the oxygen concentration in the sample solution (24) B m n , J. R Demas,J. N.Anol. Ckem. 1987,59,2780-27%. (25) Xu. W.; McDonough, R C.;Langsdotf, B.;Demas,I. N.; DeGra& B.A A n d Ckem 1994, 66,4133-4141.
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Paltial Pressure of Oxygen (mm Hg) Fipun 2. Stem-Vdmer plots of oxygen sensors wim R~(bpy)~*+siiidsiloxane of 20 mg/mL (v).Ru(phen)3*+-silica/siloxane of 32 mg/mL (B), and Ru(Ph*phen)32+/siloxaneof 0.33 mg/mL (0).
m u m4. Oxygen quenching of Ru(Phzphen)g+in oxygen sensing and 2 (0).The simulated cone 4 (m) and glucose sensing cones 1 (0) curves (-) for oxygen cone 4 and gluwsa cone 1 and for glucose cone 2 were generated using the two-site Stem-Volmer quenching model. For oxygen cone 4 and glucose cone 1, 61= 0.47, Q = 0.53, .GI= 0.0133, and & = O.CHJ14. For glucose mne 2. 61= 0.51, fm = 0.49, lGvI = 0.0131. and Km = 0.0016.
measured oxygen concentrations in the sample solution. ?he concentrationsof oxygen and glucose, therefore, can be monitored simultaneously. F i 4 shows the oxygen quenching data for one oxygen (spot 4) and two glucose (spots 1 and 2) sensing cones. ?he nonlinear curves suggest multiple quenching sites withim the polymer mahix having different sensitivities to oxygen quench ing.s Spots 1 and 4 were prepared under exactly the same conditions, and the oxygen sensitivities of both the glucose and oxygen sensing sites are virtually identical. Spot 2 was prepared using a shorter photopolymektion time. It is more sensitive to oxygen since the oxygen-sensitivedye withii the polymer matrix was exposed to less light during polymerization. Atwwite Stern-Voher quenching model was used to fit the oxygen quenching data (eq 1):=
I JI = l/&/(l+K,,[O,I) Fluorescence image of a four-spot fiber-optic sensor in pH 7.4 phosphate buffer.Spots 1 and 2 are glucose sensitive, and spots 3 and 4 are oxygen sensitive only. rlgure 3.
and the diffusion of oxygen within the polymer. At the glucose sensing sites, glucose oxidation is catalyzed by glucose oxidase within the HEMA polymer layer as glucose diffuses into the polymer matrix. Catalysis causes depletion of oxygen in the HEMA polymer, leading to depletion in the inner siloxane cone, resulting in a fluorescence intensity increase. ?he changes in fluorescence intensity are related to both glucose and oxygen concentrations in the sample solution and the diffusion of both glucose and oxygen in the polymers. Since oxygen fluctuations in the sample solution can be monitored by analyzing the fluorescenceintensity at the oxygen sensing site, the oxygen effect on the glucose sensor can be determined on the basis of the
+&/(I
+ K~IO21)) (1)
where I is the fluorescence intensity at an oxygen concentration of IO,], lois the value in the absence of quencher oxygen, fa. is the fraction of quenching site j contributing to the unquenched intensity I, and the Kw values are the Stern-Volmer quenching constants at quenching 6tej.X F i r e 4 presents the best-fitting curves, withhi = 0.47 f 0.03. MI = 0.013 f 0.015 mmH&,fm = 0.53 f 0.03, and Kn = 0.0014 f O.ooo9 mmHg-’ for glucose cone 1 and oxygen cone 4, andhi = 0.51 k 0.39,K,I = 0.013 f 0.016 “ H g - 1 , fm = 0.49 f 0.04,and K n = 0.0016 f 0.0013 “Hg-1 for glucose cone 2. In order to examine the effect of oxygen on the glucose sensor response, glucose responses were measured under different oxygen tensions with the dualanalyte sensor. ?he fluorescence intensities (0at glucose or oxygen sensing sites for differrnt concentrations of glucose and oxygen were measured. and loll values were calculated. With each value of fJ1,the corresponding quencher oxygen concentration was calculated by solving the Ana/ytica/ Chistry, Vd. 67, No. 20 October 15, 1995 3749
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Figure 5. Dual-analyte sensor responses to oxygen (open symbols) and glucose (solid symbols) with oxygen partial pressures of 76 (triangles), 159.6 (squares), and 380 (circles) mmHg.
following quadratic equation derived from eq 1:
Since the concentration of oxygen in the polymer matrix was unknown, the partial pressure of oxygen in the gas used to saturate the calibrated buffer was applied in the above equations. Figure 5 presents glucose response curves of a glucosesensitive polymer matrix (spot 1) and an oxygen-sensitive polymer matrix (spot 4) under varying oxygen tensions. At a fixed oxygen concentration,the response of the oxygen sensor is virtually constantwith increasingglucose concentration, while the response at the glucose sensor decreases due to consumption of oxygen during glucose oxidation. The difference ( P o 3 between the oxygen concentrationsin the medium and in the glucosesensitive polymer matrix corresponds to the glucose concentration. From Figure 5, the oxygen concentration withiin the glucose sensing cone drops to almost zero at a glucose concentration of 10 mM, and further increases in glucose concentration do not &ct the glucose sensor response at an oxygen tension of 76 mmHg. This result indicates that oxygen depletion inside the glucose sensing cone is complete at glucose concentrationsI 10 mM. Similarly, oxygen depletion is complete at glucose concentrations 220 mM at an oxygen tension of 159 mmHg. Thus, the dynamic range of the sensor can be extended by increasing the oxygen partial pressure. Calibration curves of glucose were made by plotting A h z versus glucose concentration (see F i e 6). For glucose sensing cone 1, a linear calibration curve is obtained with an oxygen partial pressure of 380 mmHg over the entire glucose concentration range of 0-20 mM. However, calibration curves at lower oxygen tensions are somewhat downward curved at high glucose concentrations, due to depletion of oxygen within the glucose sensing cone, as discussed above. The slopes of the calibration curves over the linear ranges for three different oxygen tensions are almost the same, indicating that sensitivity to glucose is 3750
Analytical Chemistry, Vol. 67, No. 20, October 75, 7995
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Figure 6. Glucose calibration curves at oxygen pressures of 76 (A),159.6 (W), and 380 (0)mmHg for glucose cone 1 and 159.6 mmHg (0)for glucose cone 2.
independent of the oxygen concentration outside the sensing layer. As can be seen in Figure 6, the linear ranges of the glucose calibration curves increase with increasing oxygen partial pressures. The detection limit (DL) is estimated to be 0.6 mM (S/N = 3) for glucose sensing cone 1. The glucose response can be altered by changing the amount of glucose oxidase immobilized in the HEMA matrix. Figure 6 shows a glucose calibration curve at an oxygen partial pressure of 159.6 mmHg for glucose sensing cone 2. Compared to glucose cone 1, glucose cone 2 is less sensitive (DL, 1.5 mM) because less enzyme was immobilized. Although the sensitivity of this sensor is lower, it possesses a wider dynamic range due to less oxygen depletion at a given glucose concentration. Glucose cone 2 afforded a linear glucose response curve over the glucose concentration range of 0-20 mM. This result indicates that the dynamic range of glucose response can be increased by using a lower enzyme loading. With an imaging fiber, multiple glucose sensing sites with different enzymatic activities can be placed on a single fiber surface. The sensitivity and dynamic range at a particular glucose sensing site varies according to its enzymatic activity. Therefore, any desired dynamic range of glucose response can be obtained by photodepositing the appropriate amount of glucose oxidase. The dual-analyte sensor has been used to predict glucose concentrations under different oxygen pressures. The fluorescence images of glucose and oxygen sensing polymer cones were collected with sample solutions containing different concentrations of glucose and oxygen. The steady-state fluorescenceintensities were measured and converted to correspondingPoz on the basis of the two-site quenching model. Glucose concentrations were predicted using the calibration curves of A&, versus glucose concentration. Figure 7 shows the plot of the predicted glucose concentration versus the actual glucose Concentration. The excellent correlation proves the utility of the sensor calibration scheme. The sensor was also tested for its response to interfering substances usually found in biological samples, such as ascorbic acid (AA),uric acid, and acetaminophen. These three compounds are known to be major interferents in electrochemical measure-
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ments of glucose because they are oxidized at the same potential as hydrogen peroxide. Our experimental results indicate that both saturated uric acid and acetaminophen solutions do not interfere with glucose and oxygen responses (data not shown). In biological samples,extracellular AA concentrations vary between 100 and 500 ~ M . 2 ~The 3 ~ ~sensor's response to 50 mM glucose was measured at AA concentrationswithin this range. No siffnificant interference on either oxygen or glucose sensors was observed with AA concentrations up to 2 mM (see Figure 8). Therefore, the sensor is applicable to biological measurements. AA samples with much larger concentrations were found to influence responses of both oxygen and glucose sensors because AA can be
oxidized spontaneously in aqueous solution by either oxygen or hydrogen peroxide (data not s ~ o w ~ ) . ~ ~ - ~ ~ Sensor response times and stability were also examined. F i e 9 shows the dynamic response curves of glucose monitored by injecting 0.5 mL of 1.0 M glucose solution into 5 mL of pH 7.4
(26) Grunewald, R A; O'Neil, R D.; Fillenz, M.; Albery, W. J.J. Neurochem. Inf. 1983,5,773-778. (27) Grunewald, R A Brain Res. Rev. 1993,18, 123-133.
(28) Hughes, D. E. Anal. Chem. 1985,57,555-558. (29) Mushran, S. P.;Agrawal, M. C. /. Sci. Ind. Res. 1977,36, 274-283. (30) Lowry, J. P.;O'Neil, R D. Anal. Chem. 1992,64,456-459.
Oxygen Partial Pressure (mm,Hg)
Figure 10. Stability of the dual-analyte sensor. (A) Stern-Volmer plots for oxygen sensing polymer matrix on the first (0)and second (0)days. (B) Glucose calibration curves for glucose sensing polymer matrix on the first (0)and second (0)days.
Analytical Chemistry, Vol. 67,No. 20, October 15, 1995
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phosphate buffer. The sensor response time increases with the thickness of the enzymatic polymer layer. The glucose sensor with a thicker glucose oxidase-containing poly-HEMA layer has a response time (time required to reach 95%of the steady state signal) of 28.3 f 1.7 s (three measurements), and the sensor with a thinner layer has a response time of 8.8 f 1.3 s (four measurements). The sensor recovery time is -1 min (data not shown). Figure 1OA shows the response to oxygen, and Figure 10B shows the glucose calibration curves for a sensor on two consecutive days. The fluorescenceintensity at each sensing site decreases when the sensor is exposed to excitation light because the RuOI) complex photodecomposes. However, the results in Figure 10 indicate that the sensitivity of this dual-analyte sensor to both oxygen and glucose is maintained over 2 days.
both quantitative information and spatial distribution of substrates are obtained simultaneously. In addition, a sensor with a wide dynamic range can be obtained by photodepositing multiple sensing sites with varying enzymatic activities. This compact dualanalyte sensor (350 pm 0.d.) has a fast response time (
